The present invention is directed to systems and methods for detecting and quantifying whole length or intact Fibroblast Growth Factor (FGF)-23 in a biological sample, as well as detecting and quantifying N-terminal or C-terminal fragments thereof, also present in such sample. The present invention further provides for methods for determining the relative amounts of intact FGF-23 relative to said N-terminal and C-terminal fragments.
FGF-23 is the 22nd documented fibroblast growth factor that, in humans, consists of a 251 amino acid protein having the sequence set forth at SEQ ID NO.:1. Among FGF-23's distinguishing features include a 24 amino acid signal peptide in the N-terminal portion and a unique C-terminal structure. Exemplary of the teachings of FGF-23 and its structure and properties are set forth in U.S. Pat. No. 7,223,563, issued May 29, 2007 entitled FIBROBLAST GROWTH FACTOR (FGF-23) NUCLEIC ACIDS, the teachings of which are expressly incorporated herein by reference.
FGF-23 is a major regulator of phosphate homeostasis. In this regard, when excess phosphate is present, the kidneys normally excrete such excess in the urine, and when more phosphate is needed by the body, the kidneys reabsorb the phosphate into the blood stream. With respect to the latter function, FGF-23 signals the kidneys to stop reabsorbing phosphate into the blood stream. Research has further suggested that FGF-23 helps determine how much phosphate from a person's diet is absorbed by the intestines and further plays a role in regulating the formation of biologically active Vitamin D analogs.
FGF-23 is primarily secreted by bone, followed by the thymus, heart and brain. Low levels are also secreted by other tissues. In order to function, FGF-23 must be secreted from the cells and must bind to a receptor protein. In order to be secreted, FGF-23 must be glycosylated whereby sugar molecules are attached to FGF-23 via a protein, namely, ppGalNacT3. As understood, such glycosylation allows FGF-23 to move out of the producing cell and further protects the FGF-23 molecule from being degraded. Once outside of the secreting cell, FGF-23 binds to the FGF-23 receptor protein that spans the membrane of the kidney cells whereby binding of FGF-23 to such receptor stimulates signaling that stops phosphate reabsorption by the kidneys into the blood stream.
Importantly, only “intact” FGF-23 is considered bioactive in regards to its role in phosphate regulation. In this regard, it is well understood that the first 24 amino acids of the N-terminal of FGF-23 consists of a signal peptide and that the secreted, bioactive form of FGF-23 is understood to consist of 227 amino acids extending from the 25th amino acid residue to the 251st, as set forth at SEQ ID NO.: 2. Moreover, FGF-23 is cleaved between amino acid positions 179-180, which consequently inactivates the protein and produces both an N-terminal fragment consisting of amino acids 25-179 (SEQ ID NO.: 3) and a C-terminal fragment consisting of amino acids 180-251 (SEQ ID NO.: 4). Such cleavage is understood to facilitate and regulate the amount of active FGF-23 circulating in the blood stream. Moreover, by means of a feedback loop, falling serum phosphate levels lead to diminished FGF-23 secretion thus further decreasing phosphate reabsorption.
Measurement of serum FGF-23 can assist in the diagnosis and management of disorders of phosphate and bone metabolism in patients with either normal or impaired renal function. In this respect, over-activity of FGF-23 reduces phosphate reabsorption by the kidneys, leading to low levels of phosphate in the blood (i.e., hypophosphatemia). Such conditions can cause problems with bone growth and strength, as occurs in people afflicted with autosomal dominant hypophosphatemic rickets (ADHR) and other phosphate-wasting disorders such as X-linked and autosomal recessive hypo-phosphotemia. A shortage of available FGF-23, on the other hand, decreases the signaling function of FGF-23 and consequently increases the amount of phosphate that is reabsorbed back into the blood stream by the kidneys, leading to hyperphosphatemia, which can result in calcinosis whereby excess phosphate combines with calcium to form deposits that build up in soft tissues.
Given the clinical importance of determining the presence and quantity of FGF-23, commercial immunoassays have been developed that are operative to detect and quantify the presence of FGF-23. Exemplary of such products include the Human Intact FGF-23 ELISA Kit produced by Immutopics, Inc. of San Clemente, Calif., which are disclosed and claimed in U.S. Pat. No. 7,094,551 entitled IMMUNOASSAYS, ASSAY METHODS, ANTIBODIES AND METHOD FOR CREATING ANTIBODIES FOR DETECTING FGF-23, issued on Aug. 22, 2006, the teachings of which are expressly incorporated herein by reference. Likewise, commercial immunoassays are available for detecting the intact as well as post-cleavage C-terminal fragments of FGF-23, which include the Human FGF-23 (C-Term) ELISA Kit, also produced by Immutopics, Inc. of San Clemente, Calif. With respect to the latter, the same is operative to detect the carboxyl-terminal portion of FGF-23, namely, amino acids 180-251 produced following cleavage of the whole length FGF-23 molecule, as discussed above.
Despite the importance of measuring serum FGF-23 and, in certain cases, the cleaved fragments thereof, substantial shortcomings exist with respect to the ability to not only detect the relative amounts of intact serum FGF-23 and the cleaved fragments thereof, but the relative amounts or ratios of intact FGF-23 as compared to its inactive cleaved fragments. In this regard, there are a number of specific conditions where the relative amounts of intact FGF-23 and the inactive fragments of FGF-23, and in particular the C-terminal fragments of FGF-23, have profound clinical implications. For example, in rare genetic disorders, such as mutations in GALNT3 that prevent normal glycosylation of FGF-23, only very little biologically active intact FGF-23 is secreted by osteocytes or other FGF-23 producing cells, thus often leading to severe hyperphosphatemia and consequently vascular calcifications. These patients have little or no intact FGF-23 in the circulation, but often very high levels of C-terminal FGF-23 fragments.
Similarly, patients with the McCune-Albright syndrome (activating Gs-alpha mutation) can have elevated intact FGF-23 levels leading to increased urinary phosphate excretion. However, the levels of C-terminal FGF-23 fragments are more significantly elevated than the levels of intact FGF-23, especially in those patients who have hypophosphatemia due to mosaic expression of a constitutively active Gs-alpha in bone, which consequently leads to a bigger FGF-23 C-terminal fragment to intact FGF-23 ratio.
Still further, research has indicated a link between the effect of dietary iron levels and the relative amounts of intact FGF-23 and the C-terminal fragments thereof. In one recently published study, wild-type mice on a low iron diet express FGF-23 mRNA very abundantly in bone, and these animals demonstrated a major increase in C-terminal FGF23 fragments levels, but not intact FGF-23, and they consequently did not become hypophosphatemic. Data consistent with these rodent data seem to likewise exist for healthy humans, in whom it was shown that iron levels correlate with C-terminal fragment FGF-23 levels but not with intact FGF-23 levels. In contrast, ADHR patients with an FGF-23 mutation that impairs cleavage at the RXXR site between amino acids 176 and 179, were shown to have a good correlation between iron levels and intact FGF-23 or C-terminal FGF-23 fragment levels. Furthermore, another study has shown that ferritin levels correlate with C-terminal FGF-23 fragments, but not with intact FGF-23 levels. Heavy blood losses in humans and the resulting iron deficiency therefrom, also appear to correlate with elevated C-terminal fragments of FGF-23, but not intact FGF-23 levels.
As another example, patients with earlier chronic kidney disease (CKD) stages have levels of C-terminal FGF-23 fragments that are often equally elevated as intact FGF-23 levels. As these patients approach end state renal disease (ESRD), intact FGF-23 is the predominant form of FGF-23 in the circulation. The intact FGF-23 appears to be the form of FGF-23 that has “off-target” effects resulting in poor outcome (i.e., increased mortality and accelerated loss of renal function in CKD).
As such, monitoring the amount and appearance of both intact FGF-23 in addition to the C-terminal fragments of FGF-23, as well as the relative amounts of one another, would be of considerable diagnostic importance. Indeed, the need for systems and methods to effectively monitor both the presence of intact FGF-23 and the C-terminal fragments thereof, including their relative amounts to one another, would be exceptionally useful in not only monitoring a variety of specific disease states, impaired renal function and other conditions, but for also monitoring the effectiveness of potential therapeutic agents that seek to modulate FGF-23 activity. With respect to the latter, it is conceivable that therapeutics could be developed that increase or decrease the amount of intact FGF-23 in circulation, as opposed to the cleaved inactive fragments thereof. In this regard, any type of therapeutic that is operative to accelerate the cleavage of intact FGF-23 could be beneficial for the treatment of patients with FGF-23 dependent hypophosphatemia, such as X-linked hypophosphatemia (XLH) or autosomal recessive hypophosphatemia (ARHP). Likewise, accelerated FGF-23 cleavage would be of considerable importance for patients with chronic kidney disease or end stage renal disease, who have tremendous FGF-23 elevations that are strongly expected in contributing to cardiac hypertrophy and to kidney disease progressions.
Accordingly, there is a substantial need in the art for immunoassays and methods that can serve such diagnostic needs and potential applications. There is a further need in the art for such systems and methods that are highly accurate, can be readily deployed utilizing known, existing immunoassay technology, are exceptionally effective and efficient, and are operative to provide diagnostic information, particularly with respect to ratios and relative amounts of intact FGF-23 versus fragments thereof that have not heretofore been available.